Methods and compositions for controlling plant development

ABSTRACT

A family of genes has been found in plants, said genes encoding a family of developmental proteins that have homologous structures to the mammalian QM genes. Recombinant molecules which include the QM genes in plants are useful to transform cells and regenerate plants that, as a result, have altered developmental pathways. Methods of producing male sterile plants use recombinant molecules containing either the QM sense genes or antesense genes with appropriate promoters.

This application is a divisional of application Ser. No. 08/033,797,filed Mar. 18, 1993, U.S. Pat. No. 5,383,210.

BACKGROUND OF THE INVENTION

The structure and genetic coding sequences of a family of developmentalproteins in plants have homology to mammalian QM proteins and to genesencoding the proteins. Recombinant molecules comprising plant QM codingregions and suitable promoters, are used to produce a tranformed plantwith altered development. The altered development causes male sterility.

The expression of most, if not all, plant genes can be considered to berelated in some way to plant development. Many classes of genes areknown to respond to development signals involved in celldifferentiation, formation of tissues and organs, or in controllingplant growth. There are several well-characterized examples: genes thatare regulated by light (such as rbcS and cab gene families), or byhormones and genes that express specifically in anthers, roots, seeds orleaves, or in specific cell types in these tissues (See Edwards andCoruzzi, 1990 and Kuhlmeier, Green and Chua, 1987 for reviews). Othertypes of genes are known to regulate the expression of yet other genes,such as the maize regulatory gene Opaque2 that codes for atranscriptional activator which regulates the expression of 22kd zeingenes (Schmidt et al., 1992, Ueda et al., 1992) or the C1. and R genesin maize that code for transcriptional activators that regulate theexpression of A1 and BZ1 (Klein et al., 1989).

A very new area of research in plants includes the identification andisolation of genes from plants, which, based on their homology to genesfrom animal and yeast systems, are believed to be involved in thecontrol of basic cell processes such as cell division (See Jacobs, 1992for a review). An example of such a gene is the homologue of the yeastcdc2 gene which has been cloned from maize (Colasanti, et al., 1991). Inthe future, there are certain to be additional genes identified inplants which control other basic cellular or developmental processes.

In mammals, developmental proteins have been implicated in abnormal celldivision such as characterizes the malignant state. For example, Wilms'tumor is a pediatric tumor of the kidney which arises in embryonicblastoma cells and occurs in both sporadic and hereditary forms. Threegroups have reported the cloning of two distinct genes which areassociated with Wilms' tumor. The first, WT1, encodes a zinc fingerprotein belonging to the early growth response (EGR) gene family andmaps to the 11p13 locus in humans, which is often deleted in tumorigeniccells (Call et al., 1990, Gessler et al., 1990). The second gene, termedQM, was originally cloned by Dowdy Et al. Nuc. Acids Res., 19:5763-5769, (1991) through the use of subtractive hybridization usingcDNAs and tNA derived from tumorigenic and non-tumorigenic Wilms'microcell hybrid cells, respectively. This gene was shown to beexpressed at the RNA level in virtually all normal tissues examined inthe mouse but was lacking in Wilms' tumorigenic cell lines.

The protein encoded by this gene is 25 kD in size and is very basic witha pI of approximately 12.0. Dowdy also demonstrated that QM is a memberof a family of genes in a number of mammals, particular primates. vanden Ouweland et al. (1992) cloned the QM gene from a human Xqterchromosome library and showed that this gene was 100% similar to thepreviously cloned QM gene. The expression of the QM gene has beendemonstrated in the mouse (Dowdy et al., 1991), has been cloned in thechicken, and with data from van den Ouweland et al., suggests that thisgene is conserved across a large phylogenetic range. It was postulatedthat QM may be involved in maintenance of the non-tumorigenic phenotype(Dowdy et al. 1991). It would not be expected to find the QM gene inplants, which do not have comparable phenotypes.

Discovery of genes which would alter plant development would beparticularly useful in developing genetic methods to induce the malesterility because other methods currently available have seriousshortcomings (e.g., detasseling, CMS, SI, and the like).

Production of hybrid seed for commercial sale is a large industry.Hybrid plants grown from hybrid seed benefit from the hererotic effectsof crossing two genetically distinct breeding lines. The agronomicperformance of this offspring is superior to both parents, typically inrigour, yield and uniformity. The better performance of hybrid seedvarieties compared to open-pollinated varieties makes the hybrid seedmore attractive for farmers to plant and thereby commands a premiumprice in the market.

In order to produce hybrid seed uncontaminated with self-seed,pollination control methods must be implemented to ensurecross-pollination and not self-pollination. Pollination controlmechanisms can be mechanical, chemical or genetic.

A mechanical method for hybrid seed production can be used if the plantspecies in questions has spatially separate male and female flowers orseparate male and female plants. The corn plant, for example, has pollenproducing male flowers in an inflorescence at the apex of the plant andfemale flowers in the axils of leaves along the stem. Outcrossing isassured by mechanically detasselling the female parent to preventselfing. Even though detasseling is currently used in hybrid seedproduction, the process is labor intensive and costly (yield loss isincurred).

Most major crop plants of interest, however, have both functional maleand female organs within the same flower so emasculation is not a simpleprocedure. It is possible to remove by hand the pollen forming organsbefore pollen shed, however, this form Of hybrid seed production isextremely labour intensive and, hence, expensive. Seed is produced inthis manner if the value and amount of seed recovered warrants theeffort.

A second general method of producing hybrid seed is to use chemicalsthat kill or block viable pollen formation. These chemicals, termedgametocides, are used to impart a transitory male-sterility. Commercialproduction of hybrid seed by use of gametocides is limited by theexpense and availability of the chemicals and the reliability and lengthof action of the applications. These chemicals are not effective forcrops with the extended flowering period because new flowers will beproduced that will not be affected. Repeated application of chemicals isimpractical because of costs.

Many current commercial hybrid seed production systems for field cropsrely on a genetic method of pollination control. Plants that are used asfemales either fail to make pollen, fail to shed pollen or producepollen that is biochemically unable to effect self-fertilization. Plantsthat are unable (by several different means) to self pollinatebiochemically are termed self-incompatible. Difficulties associated withthe use of self-incompatibilities include availability and propagationof the self-incompatible female line and stability of theself-compatibility. In some instances, self-incompatitability can beovercome chemically or immature buds can be pollinated by hand beforethe biochemical mechanism that blocks pollen is activated.Self-incompatible systems that can be deactivated are often veryvulnerable to stressful climatic conditions that break or reduce theeffectiveness of the biochemical block to self-pollination.

Of more widespread interest for commercial seed production are systemsof pollen control based genetic mechanisms causing male sterility. Thesesystems are of two general types: (a) genic male sterility, which is thefailure of pollen formation because of one or more nuclear genes or (b)cytoplasmic-genetic male sterility (commonly called cytoplasmic malesterility or CMS) in which pollen formation is blocked or abortedbecause of a defect in a cytoplasmic organelle (mitochondrion).

Nuclear (genic) sterility can be either dominant or recessive. Adominant sterility can only be used for hybrid seed information ifpropagation of the female line is possible (for example, via in vitroclonal propagation). A recessive sterility could be used if sterile andfertile plants are easily discriminated. Commercial utility of genicsterility systems is limited however by the expense of clonalpropagation and roguing the female rows of self-fertile plants.

Many successful hybridization schemes involve the use of CMS. In thesesystems, a specific mutation in the cytoplasmically locatedmitochondrion can, when in the proper nuclear background, lead to thefailure of mature pollen formation. In some instances, the nuclearbackground can compensate for the cytoplasmic mutation and normal pollenformation occurs. The nuclear trait that allows pollen formation inplants with CMS mitochondria is called restoration and is the propertyof specific "restorer genes" Generally, the use of CMS for commercialseed production involves the use of three breeding lines, themale-sterile line (female parent), a maintainer line which is isogenicto the male-sterile line but contains fully functional mitochondria andthe male parent line.

The male parent line may carry the specific restorer genes (usuallydesignated a restorer line) which then imparts fertility to the hybridseed. For crops, such as vegetable crops for which seed recovery fromthe hybrid is unimportant, a CMS system could be used withoutrestoration. For crops for which the fruit or seed of the hybrid is thecommercial product then the fertility of the hybrid seed must berestored by specific restorer genes in the male parent or themale-sterile hybrid must be pollinated. Pollination of non-restoredhybrids can be achieved by including with hybrids a small percentage ofmale fertile plants to effect pollination. In most species, the CMStrait is inherited maternally (because all cytoplasmic organelles areinherited from the egg cell only), which can restrict the use of thesystem. Although still used for a number of crops, limitiations of CMSsystems have a tendency to break down with prolonged use. Generally,male sterility is less than 100% effective. One particular CMS type incorn (T-cytoplasm) confers sensitivity to infection by a particularfungus.

A search for methods of altering development in plants, for example, toproduce male sterile plants, revealed an exceptionally suitable familyof developmental proteins.

SUMMARY OF THE INVENTION

The present invention relates methods and compositions for alteringplant development. The methods use genetic constructs including the QMgene in plants.

The QM gene has been described in mammals in relation to tumors, beingexpressed in normal cells, but not expressed in tumor cells. The gene islikely to be down-regulated in tumors, for example in Wilms' tumor inhumans. A gene related to mammalian oncogenesis would not be expected tohave a homologue in plants, because comparable developmentalabnormalities do not occur. Tumors are known to occur in certain plantspecies but these are specifically caused by infection by exogenousagents such as Agrobacterium or other pathogens. Yet, a polynucleotidewas isolated from the maize genome that unexpectedly showed homologywith the nucleotide sequence of the mammalian QM gene. Thatpolynucleotide from maize is referred to hereon as "QM_(m)."

The protein encoded by the maize polynucleotide is a developmentalprotein. Developmental proteins include proteins that are expressedduring development in response to a regulatory signal such as a hormone,and proteins that regulate developmental pathways. The QM gene in plantstherefore is useful in the context of controlling development, forexample, development of pollen. Interference with pollen developmentproduces a male sterile plant. Developmental proteins are recognized bytheir ability to alter the result of normal development structure orfunction.

A cDNA prepared from a QM_(m) polynucleotide consists essentially of800-950 nucleotides, including an open reading frame (0RF) and flankingregions. The comparable mammalian cDNA generally is less than 800nucleotides. The single open reading frame in the maize cDNA encodes apolypeptide of approximately 220 amino acids. In other species, an openreading frame in the QM cDNA isolated from humans, encodes a family ofQM protein of approximately 214 amino acids. In general, a QM family ofgenes in plants (QM_(p)) encodes a protein characterized by a primarysequence of approximately 200-250 amino acids, and having the followingproperties.

More specifically, genes of the QM family each encodes a family ofproteins that is characterized by the presence of three conservedregions in the amino acid sequence of the protein members of the family.In a maize QM protein, the first conserved region includes the first 20amino acids positioned from the amino terminus; the second conservedregion includes the amino acid sequence from residues 51 to 60, andforms an amphipathic helix region in the QM protein; the third conservedregion is located at residues 98-135. These three conserved regionsexhibit a high degree of homology to corresponding regions that arecharacteristic of their mammalian counterparts. "High degree ofhomology" is defined here to denote that at least 80% of the amino acidsat corresponding positions, as defined in reference to the aminoterminus of the sequence are identical.

The overall homology of a plant QM amino acid sequence, relative to amammalian counterpart, is generally at least 50%. (The differencesbetween plants and mammals occur in the region from approximatelyresidue 135 (relative to the N-terminal) to the C-terminal end of theprotein.

The nucleotide sequence positions that encode the conserved region inthe maize QM gene are located at approximately positions 30-100,positions 210-250 and positions 330-400 from the n-terminus.Hybridization probes prepared from these regions will hybridize to thecomparable mammalian QM sequences under stringent conditions.Oligonucleotide probes prepared from the conserved region are useful todetect new QM genes in plants under low stringency conditions, forexample, using 50% formamide, 5X SSC (0.75 M NaCl), at 37° C. The codingregions of the maize sequence show approximately 64% homology to thehuman QM sequence overall.

Because of "wobble" in the third position of each codon in thenucleotide sequence, a functionally similar protein would be expected tobe encoded with as much as 36% overall divergence between the nucleotidecoding regions. In the same species, a sequence encoding at least thethree conserved regions is expected to encode a functionally equivalentprotein. This is not necessarily true of cross-species comparisons,where protein function is interrelated with biochemical pathwayscharacteristic of the species. However, both the plant and the mammalianQM proteins have major effects on developmental processes.

At least two QM polynucleotides, and as many as six, are distinguishableby Northern blot analysis of maize preparations. An illustrativeembodiment of a polynucleotide encoding a QM protein is shown in FIG. 1(SEQ ID NOS 1 and 2) for maize. Oligonucleotide primers developed fromthis sequence are used to amplify the DNA in the open reading frame.These primers are useful in detection of the tobacco homologue. Theamino acid sequence corresponding to FIG. 1 (SEQ ID NO: 2) is shown inFIG. 2.

An isolated and purified plant QM protein has an estimated molecularweight of approximately 25 kD and a PI of approximately 11.0. A proteindeduced from the cDNA sequence will be free of other proteins whenprepared synthetically or by recombinant methods. Isolated and purifiedQM proteins and epitopic fragments thereof are useful in preparingantibodies. These antibodies in turn are useful for diagnosis ofdevelopmental problems and the analysis of developmental pathways inplants. The location and level of expression of the QM protein is usefulin determining how to alter development. For example, the antibodiesdeveloped to QM are useful to determine if and when the protein isturned on in specific cells or tissues of the plant. This information isuseful in developing methods for interfering with or enhancingdevelopmental pathways, including those related to pollen development.Such information is useful in developing superior plants, or malesterile plants, for example.

Isolated and purified QM proteins in plants are also useful in analyzingprotein-protein interactions. For these purposes, labeled protein probesare developed. A fusion protein including the QM protein is prepared inE. coli, for example, isolated, labelled and used in detecting proteininteractions during development. See Smith & Johnson (1988), Gene67:31-40; Ron & Dressler (1992) BioTech 13: 866-69.

A recombinant DNA molecule is prepared comprising the QM gene in thesense orientation (the orientation such that the normal mRNA istranscribed and is used as a template to translate the normal QM geneprotein) and a promoter capable of regulating transcription of said DNAin a plant cell. The recombinant DNA molecule can alternatively encodethe QM gene in the antisense orientation. This molecule contains the QMgene cloned in the opposite direction such that the minus or non-codingstrand is transcribed. No QM gene product is translated, but a MP, NAtranscript complementary to the QM mRNA is produced which is inhibitoryto the translation of the plants own QM mRNA, thus decreasing the amountof QM protein produced. (See U.S. Pat. No. 5,107,065, the contents ofwhich are incorporated by reference).

The promoter in the construct may be a cell-or tissue-specific promoter,so that the gene may be expressed in specific cells or tissues. Forexample, in a method to produce a male sterile plant, an anther specificor tapetal-specific promoter is preferred. Anther tissue and tapetalcells are examples of a tissue or cell that is crucial to development ofpollen. Anther tissue includes support cells and developing microspores,and excludes mature pollen. A QM gene construct can be effective inaltering development whether expressed in a sense or an antisenseorientation. If there are genes and processes in anther tissue which areor can be regulated by QM, a sense QM construct could affect developmentby altering the timing of regulation by QM or affect development byoverexpression of the QM protein. Correspondingly, if QM or any geneswhich can be regulated by QM are essential for normal antherdevelopment, expression of an antisense QM construct could affectdevelopment by interfering with normal QM expression.

The promoter in the construct may be an inducible promoter, so thatexpression of the sense or antisense molecule in the construct can becontrolled by exposure to the inducer. Examplary of such an inducer is aplant hormone.

Altering development is particularly useful for producing a male sterileplant. A method of producing a male sterile plant is to transform aplant cell with a recombinant molecule comprising the sense gene for theQM plant protein, or an antisense molecule directed to the QM gene. Anappropriate promoter is selected depending on the strategy fordevelopmental control. For example, a strategy is to overexpress the QMgene selectively in anther tissue by using an anther specific promoter.To produce a male sterile plant, the transformed cell would beregenerated into a plant, pursuant to conventional methodology.

A transgenic plant containing the QM gene construct can be regeneratedfrom a culture transformed with the same construct. The culture includesan aggregate of cells, a calli, or derivatives thereof that are suitablefor culture.

A plant is regenerated from a transformed cell or culture by methodsdisclosed herein that are known to those of skill in the art. Seed isobtained from the regenerated plant or from a cross between theregenerated plant and a suitable plant of the same species usingbreeding methods known to those of skill in the art.

FIG. 1 illustrates the nucleotide and derived amino acid sequence ofclone 10-15 (SEQ ID NOS 1 and 2). The cDNA clone was 936 nucleotides inlength and contained a single open reading frame encoding a polypeptideof 25,138 daltons. This polypeptide is very basic, having a calculatedpI of 11.0, with the basic residues being distributed throughout theprotein. In the search for homology with Q other previouslycharacterized genes, the amino acid sequence encoded by clone 10-15 wasused to survey the GenBank database using the TFASTA program of GeneticComputer Group (GCG, Devereux et al., 1984). This analysis yielded ascore of 716 with the human gene QM. When the amino acid sequenceencoded by clone 10-15 (SEQ ID NO: 2) was aligned with the amino acidsequence of the human gene (SEQ ID NO: 3) (FIG. 2), several regions ofinterest are seen. First, is the high degree of conservation of theamino-terminal region, where the first ten amino acid residues areconserved. The second region, again conserved in the two proteins, isbetween residues 51 and 61 which forms a putafire amphipathic helix.

The presence of a 59 residue stretch of highly conserved amino acidargues strongly for a conserved function within this region. Thecarboxy-terminal region is poorly conserved and may not be as importantin the function of the protein. Northern blot analysis of RNA isolatedfrom leaf and root tissues from seven day corn seedlings demonstratesthat cDNA from clone 10-15 is expressed in both with roots and leavesshowing roughly the same level of expression. In additional northernblots, this gene was found to be expressed in anthers and earshoots.

Southern blot analysis demonstrates that the maize homolog is a memberof a small family of approximately 4 to 6 members in maize.

METHOD OF CAUSING MALE STERILITY IN PLANTS

Use of QM Gene in Sense Orientation

The nucleotide segment of the QM gens is fused at its upstream (5') endto a promoter which is known to be specific for, or show a strongpreference for expression in, a tissue or cell that is critical forpollen development. The anther is an example of such a tissue. A tapetalcell or developing microspore is an example of a suitable cell. Thesegment is fused at its downstream (3') end to suitable transcriptionterminator and polyadenylation signals also known to function in thatcell. Preferred promoters would be SGB6 for maize and TA39 (fromtobacco) and the promoter Bp (from B. napus;) for dicots.

The present invention relates to a method for producing male sterileplants and hybrid seed, to genetic material used to impart the malesterility trait and to new products produced by said method, namely,genetically transformed plants carrying the male sterile trait, malesterile plants and hybrid seed produced by pollinating said plants withpollen from male fertile plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence and the encoded amino acid sequence of thecDNA clone 10-15 (SEQ ID NOS 1 and 3) which encodes the maize QMhomolog.

FIG. 2. Amino acid alignment analysis of the maize QM homolog (uppersequence SEQ ID NO: 2) with the human QM amino acid sequence (SEQ ID NO:3).

FIG. 3. pPHI3621 (Dp3621)

FIG. 4. pPHI1527 (Dp1527)

FIG. 5. pPHI3620 (Dp3620)

FIG. 6. pPHI3622 (Dp3522)

FIG. 7. pPHI1493 (Dp1493)

FIG. 8. pPHI4745 (DP4745)

FIG. 9. L62 Ta3914BIWT(-)!

FIG. 10. pPHI4855 (Dp4855)

FIG. 11. L59 TA39(8B3)!

FIG. 12. L61 TA39WT(AS).SEG!

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A human, constitutively expressed gene, which is thought to play a rolein maintenance of the non-tumorigenic state, has been shown to be absentin Wilms' tumorigenic cells lines. This gene, which is present as a gensfamily in humans and rodents, has been demonstrated in a number ofdiverse mammalian species. A gens has been cloned from maize whichencodes a protein having a high degree of homology with the human QMprotein (approximately 67%). The maize gene encodes a polypeptide of25kDof which basic residues comprise 22% of the protein. This gene isexpressed in all maize tissues examined by northern blot analysis and isa member of a plant gene family.

EXAMPLE 1 Interference with Normal Development of Tobacco Plants byTransformation with the QMp Gene

QMp refers to the QM gene derived from a plant. Qm_(m) refers to the QMgene derived from maize. Tobacco cells (cv. xanthi) were germinatedunder sterile conditions. After approximately 7 to 10 days under lightat 28° C., the cotyledons and first leaves were removed aseptically andcut into fourths (approximately 1-2 mm square sections) and placed ontosterile filter paper discs saturated with medium containing 0.25 Msorbitol. The discs were incubated in the dark at 28° C. overnight. Thenext morning the tissue sections were bombarded by means of abiolistical apparatus to transform cells with an equal mixture of theQM_(m) construct (pPHI3621 sense !construct or pPHI3622 antisense!construct) and plasmid containing the selectable marker (BAR gene). 0.1μg of total DNA/5 bombardments. Following bombardment, the tissue wasreturned to the 28° C. incubation in the dark. After 48 hours thebombarded tissue was transferred to selection medium (BASTA) and placedunder lights at 28° C. After about 2 weeks, small colonies began toappear, and continued to appear for about 1 week. The leaf pieces weretransferred to regeneration medium which allowed leaves and plantlets toform. After the formation of plants, the young plantlets weretransferred to rooting medium to allow root formation. After about 1-2weeks, the plants were taken to the greenhouse for planting.

The sense construct, pPHI3621 did not yield as many colonies as did thecontrol (selectable marker alone). In fact, many colonies formed yetsubsequently died. Those that lived grew at a much slower rate than thecontrols. Most of the surviving calli generated from the colonies didnot give rise to plants. Observations on the calli indicate they werehaving trouble forming or organizing a meristem to produce a plant. Mostof those that did produce plants did so from growth of a distinctportion, of the calli indicating a revertant sector (loss of theplasmid). The resulting plants were negative for the maize gene by PCRanalysis. A plant was found to be positive for the plasmid, yet didproduce a plant. However, this plant grew very slowly and did notproduce roots by the time it was transferred to the greenhouse. It grewextremely slowly in the greenhouse for some time (approximately 1 month)after which it grew at a normal rate and appeared normal. The plantflowered and set seed in a fashion similar to normal plants, however,the seeds that were produced were abnormal looking, and in germinationtests took greater than 2 weeks to germinate compared to 4-6 days fornormal seed.

The calli derived from the antisense (pPHI3622) bombarded tissues showedcompletely different growth characteristics. The calli in severalinstances grew at a much accelerated rate and produced an abundance ofvegetative growth. These calli produced plants at a near normal rate.The plantlets moved to regeneration and rooting medium produced roots ata rate faster than controls. The resulting plants appeared normal,flowered and set seed in a normal fashion. The seeds produced germinatednormally and the plants appear normal.

The QM gene plays a role in development. In transformed tobacco, mostlikely, its presence prevents or inhibits meristem formation. Whenexpressed QMs may "fix" a cell at a specific developmental stage. Afterthe gene is turned on, the cell will no longer differentiate.Overexpression of the gene in tobacco calli inhibited the formation ofmeristem to generate plants. Overexpression may be lethal at higherconcentrations.

The plant cells with constructs including the antisense molecules, wereable to grow in some cases at accelerated rates. An interpretation ofthese results is that the antisense molecule was stopping the action ofthe tobacco QM gene product, and allowing differentiation to occur morereadily and to produce the abundance of foliage seen on the calli. Theexperiment was repeated 3 times and basically the same observations weremade in each experiment.

EXAMPLE 2 Demonstration of microspore-specific gene expression by insitu hybridization

This example illustrates a method for showing that an isolated DNAcomprises a gene that exhibits microspore-specific expression. Inparticular, the results here demonstrate that expression of mRNAsrelated to a particular tobacco cDNA clone is localized to microsporesof tobacco anthers.

An anther-specific tobacco cDNA clone, designated TA39, was obtainedfrom Dr. Robert B. Goldberg of the Department of Biology, University ofCalifornia, Los Angeles, Calif. This cDNA hybridizes to mRNA fromtobacco anthers and not to mRNA from the pistil, petal, leaf or stem(Koltunow et al., 1990). The cDNA is 490 bases long, including a poly A+tail of 42 bases (SEQ. ID. NO. 1 and FIG. 1). This cDNA hybridizes totwo transcripts of 550 bases and 680 bases in Northern blots of RNAisolated from anthers. RNA dot blots have shown that TA39-relatedtranscripts accumulate and decay with the same temporal sequence as fiveother anther-specific transcripts, all of which are localized within thetaperum (Koltunow et al., 1990).

Anthers of Nicotinia tobacum (cvKy17) were collected at the tetrad stageand handled by standard cytological techniques (Berlyn and Miktha etal., 1976, BOTANICAL MICROTECHNIQUE AND CYTOCHEMISTRY, The Iowa StateUniversity Press, Ames, Iowa, Ch. 3, 4, and 5. Anthers were dehydratedin t-butanol and embedded in paraffin, then sliced into 8 μm thicksections and fixed to slides. DNA fragments of clone TA39 and anothercDNA clone (LA2: an epidermis-specific mRNA) were excised from plasmids,purified by gel electrophoresis and labeled by nick translation withbiotin-14-dATP, using the BioNick Labeling System (BRL) according todirections of the manufacturer. In situ hybridization of fixed anthersections with biotin labeled probes was carried out and detected usingthe DNA Detection System of BRL. In this system, streptavidin bindsbiotinylated probe DNA and biotinylated alkaline phosphatase, resultingin precipitation of nitroblue tetrazoliumin cells in which the probehybridizes to target nucleic acids.

Examination of these in situ hybridization analyses showed that theanther locules of the tested specimens contained tetrad stagemicrospores. In anther sections probed with TA39 DNA, only the tetradsaccumulated tetrazolium dye. In contrast, anther sections probed with acontrol DNA (LA2) accumulated dye in the epidermal layer. Thistissue-specific control demonstrates that the observed precipitation ofdye in microspores of anther sections probed by TA39 DNA is not due tononspecific retention of DNA or detection system components by themicrospores.

EXAMPLE 3 Isolation of genomic clones comprising sequences homologous tomicrospore-specific mRNA

This example provides methods of isolation of genomic DNA clonescomprising sequences homologous to any microspore-specific mRNA forwhich a nucleic acid probe is available. The approach described isuseful for isolating microspore-specific regulatory sequences from anyplant species which has microspore-specific mRNA that is homologous tosuch an available probe.

A genomic library of a selected plant, for instance a commerciallyavailable library of N. tabacum, var. NK326 DNA fragments (ClontechLaboratories, Inc., Palo Alto, Calif., catalog FL1070D), partiallydigested with MboI and cloned into the plasmid EMBL-3, was screened forclones having homology to cDNA clone TA39. Standard hybridizationmethods were used, such as are described in J. Sambrook et al.,MOLECULAR CLONING (Cold Spring Harbor Laboratory Press, 1989). Candidateclones were purified by three or more cycles of picking plaques,replating, and reprobing with a TA39 cDNA insert, until consistentlyhybridizing plaques were either purified or shown not be present.

Two distinguishable families of genomic tobacco DNA clones related tothe TA39 cDNA clone were identified, each represented by two overlappingclones within each family. One clone of each family was selected fordetailed characterization, designated clones 14B1 and 8B3. The region ofhomology with TA39 in each of these genomic clones, as well as theregions immediately upstream and downstream of these regions ofhomology, were mapped by restriction enzyme cleavage analysis and DNAhybridization.

These coding sequences and associated 5' presumptive regulatory regionswere isolated as subclones and then further subcloned for sequencing.Thus, nested sets of deletions of each genomic clone were produced byusing exoIII and mung bean nucleases supplied in a kit by Stratagene.The nested deletions were sequenced by the dideoxy chain terminationmethod of Sanger with an automated DNA sequencer (Applied Biosystems373A) at the Nucleic Acids Facility of the Iowa State University. ThecDNA insert of TA39 was also sequenced for comparison. The TA39 cDNAsequence. Within the region of homology with the TA39 cDNA of amicrospore-specific mRNA, genomic clone 8B3 is completely homologouswith TA39, while the comparable portion of genomic clone 14B1 is about90% homologous with TA39.

The starting points for transcription of the 14B1 and 8B3 genomic cloneswas mapped by primer extension experiments to a single nucleotide, 83bases upstream of the putative translational start site. A perfect TATAbox appears 31 bp upstream of the mapped start of transcription in eachclone, and a major open reading frame of 110 amino acids is intactdownstream of the start of transcription in both clones (i.e., at theposition designated "+83" relative to the transcription initiationsite). Both clones also have a polyadenylation recognition site, 29 bpand 37 bp downstream of a translational stop codon in clones 14B1 and8B3, respectively.

EXAMPLE 4 Testing for microspore-specific expression of a heterologousgene that is operatively linked to presumptive control sequences ofgenomic DNA clones

This example illustrates the use of microspore-specific regulatoryregions from genomic DNA clones to provide microspore-specific controlof expression of a heterologous reporter gene in a transient geneexpression assay.

The putative promoters of 8B3 and 14B1 were each fused to an openreading frame of a reporter gene (uidA) encoding beta-glucuronidase(GUS), followed by the 3' untranslated region of the proteinase II(pinII) gene from potato. In one version, comprising a "translational"fusion, each promoter was cloned from the beginning of the availableupstream sequences to the start of translation at nucleotide +83. Inanother variation designated a "transcriptional" fusion, each promoterwas cloned from the beginning of available upstream sequences to justbeyond the start of transcription, at nucleotide +4. The latterconstructs contained the non-translated leader of Tobacco Mosaic Virus(omega') between the promoter and uidA sequences. Translational genefusions analogous to those containing the GUS reporter gene were alsoconstructed for another model gene, the firefly luciferase codingregion.

The uidA gene fusions were tested in transient expression assays ontobacco (cv. Petite Havana) stage 3-4 anther slices bombarded by aparticle gun with DNA precipitated onto 1.8 μm tungsten beads. See, forinstance, McCormick, et al., 1991. Each shot contained 0.5 μg of DNA.Dark blue-staining Spots were observed on anther slices and inindividual microspores, indicating that transient expression of the GUSgens had occurred in microspores. The source of spots that were observedoccasionally on the anther surface could not be distinguished as towhether they arose from anther cells or stray microspores. However, inadditional tests with isolated microspores and leaves, transientexpression was confirmed for uidA and luciferase gens fusions inmicrospores. Transient assays of the luciferase constructs in leafpieces demonstrated that no gene expression activity of themicrospore-specific control sequences was observed in leaves, using themost sensitive assay available (luciferase-catalyzed luminescencedetection).

EXAMPLE 5 Preparation of genetic constructs for microspore-specificexpression of genes for insect control or male sterility

This example illustrates genetic engineering methods for producingconstructs that provide microspore-specific gene expression ofheterologous genes, such as genes that effect insect control or malesterility, in transgenic plants.

To provide constructs for microspore-specific expression of genesencoding desired proteins, for instance, a selected insect-control geneor male sterility gene, a DNA segment comprising microspore-specificregulatory sequences of this invention is operatively linked to aheterologous gene, and to 3'-non-translated. sequences, as needed, forproviding translational and transcriptional control appropriate for theselected heterologous gene. The regulatory sequences are fused withheterologous gene sequences, for example, by modifying the beginning ofthe open reading frame of the heterologous gene to include a restrictionenzyme cleavage site. Advantageously, this cleavage site is an NcoI siteor another site compatible for ligation with an NcoI site, because thesequences of such sites comprise an ATG translation start codon.

A variety of genotypes were used for this example wherein xanthi tobaccotransformations were performed at the 10 day germination stage.

    ______________________________________                                        The constructs are described as follows:                                      ______________________________________                                        pPHI3621 + pPHI1285  QM, maize sense + BAR!                                   pPHI3622 + pPHI1285  QM, maize antisense + BAR!                               pPHI4722 + pPHI1285  QM, human sense + BAR!                                   pPHI4723 + pPHI1285  QM, human antisense + BAR!                               pPHI4280 + pPHI1285  QMUS + BAR!                                              pPHI265 + pPHI1285  GUS + BAR!                                                pPHI1285  BAR!                                                                ______________________________________                                    

To achieve transformation, a particle gun bombardment was used, a GEHelium gun and 650PSI rupture disks.

One bombardment was done per sample, for a total of 0.1 μg.

Tobacco was germinated and observed in vitro on 272 medium for 10-14days before the following steps. One day before the experiment,cotyledons and first leaves were cut into halves and placed on sterilefilters containing 1.5ml of 530 medium+0.25M sorbitol. Incubation wasdone at 280° C. in the dark overnight. Leaf material was dissected underliquid medium to prevent desiccation. Eight leaf sections per plate werecultured, 5 plates were prepared per QM transformation, and 3 plateswere prepared per control transformation.

Following bombardment, all samples were maintained on the originalfilters for 2 days before transferring them to selection medium

After 48 hours, tissue was transferred to 526+Basta (526H) medium,leaving leaf tissue on the filters. Colony recovery generally occurredat 2-3 weeks post bombardment.

After 4 weeks, cotyledons/colonies were transferred to 528S medium.Plantlets from transformed colonies were cut off of the base callus andtransferred to 272N medium to allow for root formation to occur. Whenroots were well established, plants were transferred to greenhouse formaturing.

The results were as follows:

126 colonies were recovered from all DNA treatments this study. PCRanalysis was completed on 50 total colonies by randomly sampling 12 fromeach of the DNA treatments. Data from this analysis are shown below:

    ______________________________________                                        DNA Treatment                                                                              Percent PCR                                                                             +     Percent Plant Recovery                           ______________________________________                                        pPHI3621/pPHI1285                                                                            90%           14.3%                                            pPHI3622/pPHI1285                                                                          62.5%             20%                                            pPHI4722/pPHI1285                                                                          66.7%           16.6%                                            pPHI4723/pPHI1285                                                                            75%           28.6%                                            pPHI4280/pPHI1285                                                                          *n/a            n/a                                              pPHI265/pPHI1285                                                                            100%            100%                                            ______________________________________                                    

pPHI4280/pPHI1285 was sampled for PCR and analyzed, however, due to anendogenous tobacco sequence that was amplified with the primers, nofurther analysis or plant maturation was completed.

Differences in growth rates were observed at 6 weeks post bombardment.The observation most notable was that colonies recovered fromtransformations with pPHI3621/pPHI1285 and pPHI3622/1285 showedestablished colony death, especially from the pPHI3622/pPHI1285treatment. No noticeable differences in growth were noted for the othertransformations when compared to the control, pPHI265/pPHI1285 colonies.

EXAMPLE 6 Stable BMS transformation to evaluate the effect andexpression of QM gene in sense and antisense orientation and in theGRP/GRE inducible gene system.

The genotype used was BMS P-38 maize suspensions.

DNA constructs were:

    ______________________________________                                        pPHI4719 + pPHI1285  35S-QM sense + 35S-BAR!                                  pPHI4720 + pPHI1285  35S-QM sense + 35S-BAR!                                  pPHI4718 + pPHI4740 + pPHI1285                                                 NOS-GRP + GRE-QM sense + 35S-BAR!                                            DP1285  35S-BAR!                                                              ______________________________________                                    

Particle gun bombardment was used (a GE helium gun, and 650PSI ruptureddisks).

One bombardment was done per sample.

One day after subculture, liquid was vacuumed off the cells and 2 gramsof material was resuspended in 20 ml 237+0.25M sorbitol medium.

Cells were incubated at 28° C. on shaker apparatus for 2-4 hours.

0.5 ml of cells were plated onto double layers of Whatman filtersmoistened with 1.5 ml 237+0.25M sorbitol medium. The cell density perplate was about 50 mg.

6 samples were completed for each DNA treatment, including 2 samples asunshot controls.

Following bombardment, filters with cells were transferred to 115 mediumand returned to the dark at 28° C. for 48 hours.

Cells were transferred to 306E selection medium after 48 hours byscraping the cells off the filter, resuspending them in 2 ml of 237medium, and plating them in 1 ml per plate for each sample.

Colony recovery was monitored. When a colony was identified, it wasseparated from the others to maintain identity.

Induction assays may be performed after PCR analyses confirms presenceof genes in transgenic colonies.

Colony recovery occurred from all transformations in this example,however, the majority of recovery came from the DP1285 positive controltreatment. Data for colony recovery is shown below:

    ______________________________________                                        DNA Treatment     *N    Colonies Recovered                                    ______________________________________                                        pPHI4719/DP1285    9     3                                                    pPHI4720/DP1285   11     4                                                    pPHI4718/DP4740/DP1285                                                                          11    22                                                    pPHI1285          11    44                                                    ______________________________________                                         *N denotes the number of samples bombarded per DNA treatment.            

Both the 35S sense and antisense constructs for the QM gene were toxicto BMS colony recovery.

EXAMPLE 7 Using a Maize Tapetum Specific Promoter for Transformation

Experiment Protocols

Repetition 1, 2, and 5:

Goal: Recover transgenic colonies, plants and progeny of maize resistantto Basta/Bialaphos and expressing GUS driven by the taperum specificSGB6gl promoter.

Genotype: 54-68-5 B1-1 (Repetition 1) or 54-68-5 161F3 (Repetition2)54-68-5 161F4 (Repetition 5)

Medium: 237 liquid suspension medium for maize

115, callus maintenance medium for maize

115E, callus 5 mg/n Basta selection medium

115B, callus 3 mg/L Bialaphos selection medium

Tissue Treatment:

Sieve cells through 710 um mesh one day after subculture

Resuspend in 237+3% PEG at 50 mg/ml plate density

Incubate in 3% PEG overnight

Plate cells, 0.5 ml/plate onto glass filters 934-AH atop a Whatmanfilter moistened with lml 237+3% PEG medium

Transfer cells on glass filter to 115 medium following bombardment

Particle gun bombardment:

DuPont helium gun (Repetitions 1 and 5)

650 PSI rupture disks (Repetitions 1 and 5)

DuPont PDS-1000 gun (Repetition 2)

0.230" stopping plates, Acetyl macroprojectiles (Repetition 2)

One bombardment per sample (Repetitions 1 and 5)

Two bombardments per sample (Repetition 2)

Pioneer tungsten modified DNA protocols, specific to each gun

DNA:

pPHI687+DP610

pPHI460+DP610

pPHI1952+DP610

pPHI2125+DP610

Treatment/Assay following bombardment:

Look for R gene expression 24-48 hours post bombardment

Transfer samples to 115E (repetitions 1) 48 hours post bombardment.Transfer samples to 115B (repetition 2 and 5) 7 days post bombardment

Transfer cells off filters 2 weeks following transfer to selection

PCR assay colonies for reporter gene prior to plant regeneration

Maintain samples at 28° C. in the dark

Repetition 1

PCR assays were completed on 16 independent colonies recovered on 5mg/LBasta selection. One colony, #9 plate 1CZ, DP610+DP2125 was PCR positivefor GUS (DP2125). All colonies were Type I phenotypes--however, thenonselected positive control also became a Type I phenotype. Thisphenotype tends to be common in the 54-68-5 B1-1 line. After 12 weeks on5 mg/L Basta selection, all PCR negative colonies were discarded alongwith all remaining nonembryogenic tissue. Colony 2 from Sample #9 plate1 was transferred to 288E (Regeneration medium+5mg/L Basta). Eightcolonies remained to be PCR assayed for the presence of the GUS gene. Ofthese eight colonies, three were PCR positive for GUS from either thetranslational fusion (DP2125) or the transcriptional fusion (DP1952).

Repetition 5

PCR assays were completed on nine independent colonies recovered on 3mg/L Bialaphos selection. All colonies were PCR positive for the GUSgene, indicating the presence of either DP2125 or DP1952. Gene controlsused in this experiment (DP460) have yielded 9 stable transformants, allof which have areas that stain blue in a GUS cytochemical assay. Growthwas much faster in the gene controls than in the transgenics recoveredfrom the SGB6gl:GUS constructs.

After 12 weeks under selection pressure, only fast growing, embryogeniccolonies were kept--all other material being discarded. Colonies testingPCR positive were transferred to regeneration medium for plant recovery.Basta enzyme assays were completed on a portion of the colonies. Resultsshown in the data table do not indicate a high degree of transgenicsactively showing resistance to Basta. From previous work and otherresearchers' experiences with this assay, a more reliable measure oftransformation has become looking for the cell morphology of therecovered colonies to closely resemble that of the nonselected controlsplus the rate of growth the recovered colonies exhibit.

Tungsten/DNA Protocl for DuPont Helium Gun

Weigh 60 mg 1.8 μm tungsten: put into 15 ml centrifuge tube

Add 2 ml 0.1M HnO₃ : Sonicate on ice for 20 minutes

Withdraw HNO₃ : Add 1 ml sterile deionized water and transfer sample toa 2 ml Sarstedt tube. Sonicate briefly Centrifuge to pellet particles

Withdraw H₂ O: Add 1 ml 100% EtOH--Sonicate briefly Centrifuge to pelletparticles

Withdraw H₂ O: Add 1 ml 100% EtOH--Sonicate briefly Centrifuge to pelletparticles

Withdraw EtOH. Add 1 ml sterile deionized water. Sonicate.

Pipet 250 μl of suspension into 4, 2 ml tubes.

Add 750 μl of sterile deionized H₂ O to each tube.

Freeze tungsten sample between use.

To prepare DNA

Piper 50 μl tungsten/H₂ O suspension into 1.5 ml tube (Sonicate first)

Add 10 μg DNA, Mix

Add 50 μl 2.5M CaCl₂. Mix

Add 20 μl 0.1M Spermidine. Mix

Sonicate briefly. Centrifuge for 10 seconds at 10,000 RPM.

Withdraw supernatent. Add 250 μl 100% EtOH. Sonicate briefly.

Centrifuge at 10,000 RPM for 10 seconds

Withdraw supernatent. Add 60 μl 100% EtOH.

EXAMPLE 8 Construction of Plasmids Containing The the Maize QM Gene

Plasmid pPHI3621 (FIG. 3) which expresses the QM gene (WT2H) in thesense orientation was constructed using pPHI1527 as one parent. pPHI1527(FIG. 4) contains the plasmid pUC18 as the backbone (Yanisch-Perron, C.,Vieira, J. and Messing J., 1985 , "Improved M13 cloning vectors and hoststrains: Nucleotide sequences of the M13mp18 and pUC19 vectors." Gene33:103-119) which contains the restriction sites necessary for cloningand the ampicillin resistance gene as a selectable marker. It alsocontains the cauliflower mosaic virus (CaMV) 35S promoter and enhancersequences (Gardner, R. C., Howarth, A. J., Hahn, P., Brown-Luedi, M.,Shepherd, R. J. and Messing, J. C., 1981, "The complete nucleotidesequence of an infectious clone of cauliflower mosaic virus by M13mp7shotgun sequencing." Nucleic Acids Res. 9:2871-2888), the tobacco mosaicvirus leader sequences, O', (Gallie, D. R., Slex, D. E., Watts, J. W.,Turner, P. C. and Wilson, T. M. A., 1987, "The 5' leader sequence oftobacco mosaic virus RNA enhances the expression of foreign genetranscripts in vitro and in vivo." Nucleic Acids Res. 8:3257-3273), thefirefly luciferase reporter gene (OW, D., Wood, K. V., DeLuca, M., deWet, J. R., Helinski, D. R., and Howell, S. H., 1986, "Transient andstable expression of the firefly luciferae gene in plant cells andtransgenic plants." Science 234:856-859) and the PinII transcriptionterminator sequences (Hynheung, A., Mitra, A., Choi, H. K., Costa, M.A., An, K., Thornburg, R. W. and Ryan, C. A., 1989, "Functional analysisof the 3' control region of the potato wound-inducible proteinaseinhibitor II gene." Plant Cell 1:115-122). The second parent of pPHI3621was pPHI3520, which pBluescript KS- containing the maize QM gene (FIG.5). pPHI3621 was generated by digestion of both pPHI3620 and pPHI1527with NcoI and KpnI and isolation of the insert band from pPHI3620 andthe larger plasmid band from pPHI1527 on low melting point (LMP) agarosegels. This strategy replaced the luciferase gene with the maize QM gene.The bands were pooled and ligated to form pPHI3621.

pPHI3622 (FIG. 6), which expresses the antisense of the maize QM genewas constructed using pPHI1527 and pPHI3620 as parents, but by digestionof both with SalI and SacI. The insert band from pPHI3620 and the largerplasmid band from pPHI1527 was isolated from LMP agarose gels, thefragments were pooled and ligated. Again, this replaced the luciferasegene with maize QM gene in the antisense orientation.

Tissue specific expression vectors were constructed in the same mannerexcept that the CaMV constitutive promoter was replaced with the TA39anther specific promoters, 14B1 and 8B3 (Garnaat, C W. and Huffman, G.,1991, "Isolation and transient assay of tobacco anther specificpromoters." Abstracts: The International Society for Plant MolecularBiology. Tucson., Ariz., October 1991). pPHI1493 (FIG. 7) containing the14B1 promoter was digested with NcoI and NsiI as was pPHI3621 (parent2). The small insert band from pPHI3621 and the larger plasmid band wereisolated by LMP agarose gel electrophoresis, were pooled and ligated.This yielded pPHI4745 (FIG. 8) which contained the maize QM gene in theantisense orientation with the 14B1 promoter. The maize QM antisenseconstruct was made by digestion of pPHI4745 with SmaI and NsiI anddigestion of pPHI3622 with Sali (which was filled in with Klenowfragment) and NsiI. The large plasmid band from pPHI4745 and the insertband from pPEI3622 were isolated by LMP gel, pooled and ligated. Thisyielded the plasmid L62 (FIG. 9).

The expression vectors containing the 8B3 anther specific promoter wereconstructed by digestion of pPHI4855 (FIG. 10) with BamHI and NotI.pPHI4855 contained all of the above described sequences with theadditional sequences encoding the B-glucuronidase gene (Walden, R. andSchell, J., 1990, "Techniques in plant molecular biology-progress andproblems." Eur. J. Biochem. 192:563-576). The other parent, pPHI4745 wasalso digested with BamHI and NotI. The large plasmid band from thepPHI4855 and the insert band from pPHI4745 were purified from LMPagarose, pooled and ligated. The resulting plasmid, L59 (FIG. 11)contained the maize QM in the sense orientation driven by the antherspecific promoter 8B3. The antisense construct was made by digestion ofpPHI4855 with SmaI and NsiI and pPHI3622 with SalI (then filled in withKlenow fragment) and NsiI. The large plasmid band from pPHI4855 and theinsert band from pPHI3622 were isolated from LMP agarose gel, pooled andligated. This gave L61 (FIG. 12) the antisense orientation of the maizeQM gene behind the 8B3 promoter.

Materials and Methods

Use of QM Gene in Sense Orientation

The nucleotides segment of the QM gene isolated from maize or otherplant sources is fused at its upstream (5') end to a promoter whichallows expression of the sense gene in a particular target plant celland is fused at its downstream (3') end to suitable transcriptionterminator and polyadenylation signals known to function in that cell.Preferred promoters include those that are known to direct expression inthe desired target cell, which includes "constitutive" promoters such as35S from CaMV and the promoter from the ubiquitin gene that are known todirect expression in a wide variety of plant cell types. 35S is likelyto direct expression in both monocots such as corn and dicots such astobacco and canola. However, the ubiquitin promoter for tobaccopreferably is derived from a dicot source. The ubiquitin promoter foruse in monocots such as corn preferably is derived from a monocotsource. Other suitable promoters include those which are known to beinducible under specific conditions, such as in response to particularchemical treatments for example, an herbicide.

Terminator/polyadenylation signals include those that are known tofunction in the target cell of interest. Preferred are signals fromgenes such as pinll (proteinase inhibitor II from potato) or T-DNA genessuch as OCS or NOS, which are known to function in a wide variety ofplant cell types, including those of dicots and monocots such as corn.When the target cell is from a monocot like corn, it is preferred, butnot necessarily required, that an intron from a monocot gene be insertedbetween the promoter and the QM gene. Examples would be an intron (suchas intron 1 or 6) from the Adhl gene of corn.

Use of Maize QM Gene in Antisense Orientation

The antisense form of the QM gene is fused at its upstream (5') end to apromoter which directs expression in a particular target plant cell, andis fused at its downstream (3') end to suitable transcription terminatorand polyadenylation signals also known to function in that cell. Anembodiment of a target cell in this case is a cell in which the QM geneor a gene highly homologous to the QM gene is known to be expressed sothat the antisense works effectively. Preferred promoters encompassthose that are known to direct expression in the desired target cell,suitable candidates include "constitutive" promoters such as 35S and thepromoter from the ubiquitin gene that are known to direct expression ina wide variety of plant cell types. 35S is expected to express in bothmonocots such as corn and dicots such as tobacco and canola. However,the ubiquitin promoter for tobacco is preferably from a dicot source,and the ubiquitin promoter for use in monocots such as corn ispreferably from a monocot source. Other preferred promoters includethose which are known to be inducible under specific conditions, such asin response to a particular chemical treatment for example, a herbicide.It is preferred that the antisense construct include the entire QM geneor at least several hundred nucleotides from the 5' end of the gene.

Use of QM Gene in Antisense Orientation

The nucleotide segment of the antisense form of the QM gene is fused atits upstream (5') end to a promoter which is known to be specific for,or show a strong preference for expression in, a tissue or cell criticalfor pollen development. An example of a suitable tissue is the anther.An example of a suitable cell is a tapetal cell or a developingmicrospore. The segment is fused at its downstream (3') end to suitabletranscription terminator and polyadenylation signals also known tofunction in the cell or tissue. The target cell is a cell in which theQM gene or a gene highly homologous to the QM gene is known to directexpression so that the antisense works effectively.

TRANSFORMATION METHODS: Transformation methods for dicots include anumber of different well-known methods for direct DNA delivery.Preferred is particle biolistics bombardment of leaf explants. Othermethods include Agrobacterium delivery to explants; Agrobacteriumcocultivation of protoplasts; electroporation, PEG uptake or otherdirect DNA delivery into protoplasts, and the like. A preferred methodfor monocots such as corn is delivery of DNA to the treated cells bybombardment, but other methods such as electroporation can also be used.

Cells of a plant are transformed with the foreign DNA sequence of thisinvention in a conventional manner. If the plant to be transformed issusceptible to Agrobacterium infections, it is preferred to use a vectorcontaining the foreign DNA sequence, which is a disarmed Ti-plasmid. Thetransformation can be carried out using procedures described, forexample, in EP 0,116,718 and EP 0,270,822. Preferred Ti-plasmid vectorscontain the foreign DNA sequence between the border sequences, or atleast located upstream of the right border sequence. Of course, othertypes of vectors can be used for transforming the plant cell, usingprocedures such as direct gene transfer (as described for example in EP0,2370,356, PCT publication W0/85/01856 and EP 0,275,069), in vitroprotoplast transformation (as described for example in U.S. Pat. No.4,684,611), plant virus-mediated transformation (as described forexample in EP 0,067,553 and U.S. Pat. No. 4,407,956) andliposome-mediated transformation (as described for example in U.S. Pat.No. 4,536,475).

If the plant to be transformed is corn, recently developedtransformation methods are suitable such as the methods described forcertain lines of corn by Fromm et al. (1990) Bio/Technology 8: and 833and Gordon-Kamm et al. (1990) The Plant Cell 2: 603.

If the plant to be transformed is rice, recently developedtransformation methods can be used such as the methods described forcertain lines of rice by Shimamoto et al. (1990) Nature 338: 274, Dattaet al. (1990) Bio/Technology 8, 736, Christou et al. (1991)Bio/Technology 9, 957 and Lee et al. (1991) PNAS 88:6389.

If the plant to be transformed is wheat, a method analogous to thosedescribed above for corn or rice can be used. Preferably for thetransformation of a monocotyledonous plant, particularly a cereal suchas rice, corn or wheat, a method of direct DNA transfer, such as amethod of biolistic transformation or electroporation, is used. Whenusing such a direct transfer method, it is preferred to minimize the DNAthat is transferred so that essentially only the DNA sequence of thisinvention, the QM maize gene, is integrated into the plant genome. Inthis regard, when a DNA sequence of this invention is constructed andmultiplied in a plasmid in a bacterial host organism, it is preferredthat, prior to transformation of a plant with the DNA sequence, plasmidsequences that are required for propagation in the bacterial hostorganism, such as on origin of replication, an antibiotic resistancegene for selection of the host organism, and the like, be separated fromthe parts of the plasmid that contain the foreign DNA sequence.

PROTOCOL FOR CORN TRANSFORMATION TO RECOVER STABLE TRANSGENIC PLANTS

Day--1 Cells places in liquid media and slaved (710 um). 100-200 mg ofcells collected on 5.5 cm glass fiber filter over an area of 3.5 cm.Cells transferred to media and incubated media overnight.

Day--8 Filter and cells removed from media, dried and bombarded. Filterand cells placed back on media.

Day--5 Cells on filter transferred to selection media (3 mg bialophos).

Day--12 Cells on filter transferred to fresh selection media.

Day--19 Cells scraped from filter and dispersed in 5 ml of selectionmedia containing 8.6% low melting point sea aqarose. Cells and mediaspread over the surface of two 100 mm×15 mm plates containing 20 ml ofgel-rite solidified media.

Day--40 Putative transformants picked from plate.

Day--61 Plates checked for new colonies.

RNA ANALYSIS: Total cellular RNA was prepared from B73 seedlings sevendays following planting by the protocol of Chomczynski and Sacchi(1987). Poly (A)+ RNA was purified from leaf homogenates using thePolyAtract 1000 system (Promega). Northern blots were done as previouslydescribed (Thomas, 1980).

REFERENCES

1. Call et al., , 1990, "Isolation and characterization of a zinc fingerpolypeptide gene at the human chromosome 11 Wilms' tumor locus". Cell,60:509-520.

2. Chomczynski et al., 1987, "Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction." Anal.Biochem., 162:156-159.

3. Colasanti, J. et al., (1991), "Isolation and characterization of cDNAclones encoding a functional p34cdc2 homologue from Zea mays", Proc.Natl. Acad. Sci. USA 88:3377-3381

4. Devereux et al., 1984, "A comprehensive set of sequence analysisprograms for the VAX." Nucleic Acids Res. 12:387-395.

5. Edwards et al., (1990) , "Cell-specific gene expression in plants."Ann. Rev. Genet 24: 275-303.

6. Gessler et al., 1990, "Homozygous deletion in Wilms' rumours of azinc-finger gene identified by chromosome jumping." Nature, 343:774-778.

7. Klein et al., (1989), "Regulation of anthocyanin biosynthetic genesintroduced into intact maize tissues by microprojectiles." Proc. Natl.Acad. Sci. USA 86: 6681-6685.

8. Kuhlmeier et al., (1987), "Regulation of gene expression in higherplants." Ann Rev. Plant Physiol. 38:221-257.

9. Schmidt et al., (1992), "Opaque2 is a transcriptional activator thatrecognizes a specific target site in 22-kd zein genes." Plant Cell4:689-700.

10. Thomas et al., 1980, "Hybridization of denatured RNA and small DNAfragments transferred to nitro-cellulose." Proc. Natl. Acad. Sci. USA.,77:5201-5205.

11. Ueda et al., (1992), "Mutations of the 22- and 27-kd zein promotersaffect transactivation by the Opaque2 protein." Plant Cell 4:701-709.

12. van den Ouweland et al., 1992, "Identification and characterizationof a new gene in the human Xq28 region". Human Mol. Genet.,

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 936 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 42..704                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCGCCGACACCGACTGCCTACCTCAGCTGCCGTCGCCATGGGCAGAAGG53                       MetGlyArgArg                                                                  CCTGCTAGATGCTATCGCCAGATCAAGAACAAGCCGTGCCCTAAGTCC101                           ProAlaArgCysTyrArgGlnIleLysAsnLysProCysProLysSer                              5101520                                                                       AGGTACTGCCGTGGTGTCCCTGACCCCAAGATCAGGATCTACGATGTC149                           ArgTyrCysArgGlyValProAspProLysIleArgIleTyrAspVal                              253035                                                                        GGGATGAAGAGGAAGGGTGTTGATGAGTTCCCCTACTGTGTGCACCTT197                           GlyMetLysArgLysGlyValAspGluPheProTyrCysValHisLeu                              404550                                                                        GTCTCTTGGGAGAGGGAGAATGTCTCCAGTGAGGCGCTCGAGGCTGCC245                           ValSerTrpGluArgGluAsnValSerSerGluAlaLeuGluAlaAla                              556065                                                                        CGCATTGTCTGTAACAAGTACATGACCAAGTCTGCAGGAAAGGATGCC293                           ArgIleValCysAsnLysTyrMetThrLysSerAlaGlyLysAspAla                              707580                                                                        TTCCACCTTAGGGTCCGGGTTCACCCGTTCCATGTCCTCCGTATCAAC341                           PheHisLeuArgValArgValHisProPheHisValLeuArgIleAsn                              859095100                                                                     AAGATGCTTTCCTGTGCCGGGGCTGATAGGCTCCAGACTGGAATGAGG389                           LysMetLeuSerCysAlaGlyAlaAspArgLeuGlnThrGlyMetArg                              105110115                                                                     GGTGCCTTTGGCAAGCCTCAGGGCACCTGTGCTAGGGTGGACATTGGT437                           GlyAlaPheGlyLysProGlnGlyThrCysAlaArgValAspIleGly                              120125130                                                                     CAGGTCCTCCTTTCCGTGCGCTGCAAGGACAACAATGCTGCCCATGCC485                           GlnValLeuLeuSerValArgCysLysAspAsnAsnAlaAlaHisAla                              135140145                                                                     AGCGAAGCTCTGCGTCGCGCTAAGTTCAAGTTCCCTGCCCGCCAGAAG533                           SerGluAlaLeuArgArgAlaLysPheLysPheProAlaArgGlnLys                              150155160                                                                     ATCATTGAGAGCAGAAAGTGGGGCTTCACCAAGTTCAGCCGCGCTGAC581                           IleIleGluSerArgLysTrpGlyPheThrLysPheSerArgAlaAsp                              165170175180                                                                  TACCTGAAGTACAAGAGCGAGGGCAGAATTGTTCCTGATGGTGTCAAC629                           TyrLeuLysTyrLysSerGluGlyArgIleValProAspGlyValAsn                              185190195                                                                     GCAAAGCTGCTCGCCAACCACGGCAGACTTGAGAAGCGTGCTCCTGGG677                           AlaLysLeuLeuAlaAsnHisGlyArgLeuGluLysArgAlaProGly                              200205210                                                                     AAGGCTTTCCTCGATGCCGTTGCTTAAGTGCGGATGCGAATCCTGACGTTTTGC731                     LysAlaPheLeuAspAlaValAla                                                      215220                                                                        TTTAGCGTATCTTACTTTGCTTCGTGGAACATGAATTTCAAGTGTTTTGAGGGTATTACA791               GTGCCTTATGTGAACTTGCCTATCTTGTGCTGAACATCGGAATGTATCCTCCGAGTATGT851               TTAATCGCATTAATTTTATTGGGAAATTGGTTGCGGAACAATGTCCAATTTAACTCGAAT911               TTGATTTCAACACGGTCTTTTCTTT936                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 220 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyArgArgProAlaArgCysTyrArgGlnIleLysAsnLysPro                              151015                                                                        CysProLysSerArgTyrCysArgGlyValProAspProLysIleArg                              202530                                                                        IleTyrAspValGlyMetLysArgLysGlyValAspGluPheProTyr                              354045                                                                        CysValHisLeuValSerTrpGluArgGluAsnValSerSerGluAla                              505560                                                                        LeuGluAlaAlaArgIleValCysAsnLysTyrMetThrLysSerAla                              65707580                                                                      GlyLysAspAlaPheHisLeuArgValArgValHisProPheHisVal                              859095                                                                        LeuArgIleAsnLysMetLeuSerCysAlaGlyAlaAspArgLeuGln                              100105110                                                                     ThrGlyMetArgGlyAlaPheGlyLysProGlnGlyThrCysAlaArg                              115120125                                                                     ValAspIleGlyGlnValLeuLeuSerValArgCysLysAspAsnAsn                              130135140                                                                     AlaAlaHisAlaSerGluAlaLeuArgArgAlaLysPheLysPhePro                              145150155160                                                                  AlaArgGlnLysIleIleGluSerArgLysTrpGlyPheThrLysPhe                              165170175                                                                     SerArgAlaAspTyrLeuLysTyrLysSerGluGlyArgIleValPro                              180185190                                                                     AspGlyValAsnAlaLysLeuLeuAlaAsnHisGlyArgLeuGluLys                              195200205                                                                     ArgAlaProGlyLysAlaPheLeuAspAlaValAla                                          210215220                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 214 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetGlyArgArgProAlaArgCysTyrArgTyrCysLysAsnLysPro                              151015                                                                        TyrProLysSerArgPheCysArgGlyValProAspAlaLysIleArg                              202530                                                                        IlePheAspLeuGlyArgLysLysAlaLysValAspGluPheProLeu                              354045                                                                        CysGlyHisMetValSerAspGluTyrGluGlnLeuSerSerGluAla                              505560                                                                        LeuGluAlaAlaArgIleCysAlaAsnLysTyrMetValLysSerCys                              65707580                                                                      GlyLysAspGlyPheHisIleArgValArgLeuHisProPheHisVal                              859095                                                                        IleArgIleAsnLysMetLeuSerCysAlaGlyAlaAspArgLeuGln                              100105110                                                                     ThrGlyMetArgGlyAlaPheGlyLysProGlnGlyThrValAlaArg                              115120125                                                                     ValHisIleGlyGlnValIleMetSerIleArgThrLysLeuGlnAsn                              130135140                                                                     LysGluHisValIleGluAlaLeuArgArgAlaLysPheLysPhePro                              145150155160                                                                  GlyArgGlnLysIleHisIleSerLysLysTrpGlyPheThrLysPhe                              165170175                                                                     AsnAlaAspGluPheGluAspMetValAlaGluLysArgLeuIlePro                              180185190                                                                     AspGlyCysGlyValLysTyrIleProSerArgGlyProLeuAspLys                              195200205                                                                     TrpArgAlaLeuHisSer                                                            210                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGGGCAGAAGGCCTGCTAGATGC24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAACGGCATCGAGGAAAGCCTTCC24                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified nucleic acid thesequence of which is selected from the group consisting of:a) thenucleic acid sequence of SEQ ID NO: 1; b) a nucleic acid sequence fullycomplementary to SEQ ID NO: 1; c) an oligonucleotide comprising 10-50contiguous nucleotides of the nucleic acid sequences in a) or b) whichis capable of performing in a amplification reaction wherein a portionof a plant QM gene is selectively amplified; and d) a nucleic acid ofa), b), or c) wherein the T bases are U bases.
 2. The oligonucleotideprimers of claim 1, wherein said primers consist of the nucleotidesequence:Left (SEQ ID NO: 4): 5'-ATGGGCAGAAGGCCTGCTAGATGC Right (SEQ IDNO: 5): 5'-CAACGGCATCGAGGAAAGCCTTCC.